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Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

Hernández H, Makarova OV, Makarov EM, Morgner N, Muto Y, Krummel DP, Robinson CV - PLoS ONE (2009)

Bottom Line: Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle.Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo.These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

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A model of interactions between the C-terminal tails of U1-70k isoforms 1 (left) and 2 (right) with U1-C and Sm-B/B', respectively.Possible path of the C-terminal tails of two U1-70k isoforms drawn approximately to scale (dashed curved orange lines) and modeled onto the crystal structure of human U1 snRNP [17] and interactions between C-terminal tails of U1-C and SmB/B' are highlighted (red and blue boxes respectively). Indicated are: U1 snRNA (magenta); Sm-B/B' (blue); Sm-D1, D2, D3, E, F and G (cyan); U1-70k (orange); U1-C (red); speculative path of the C-terminal tails of U1-70k isoforms 1 and 2 (dashed orange lines).
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pone-0007202-g006: A model of interactions between the C-terminal tails of U1-70k isoforms 1 (left) and 2 (right) with U1-C and Sm-B/B', respectively.Possible path of the C-terminal tails of two U1-70k isoforms drawn approximately to scale (dashed curved orange lines) and modeled onto the crystal structure of human U1 snRNP [17] and interactions between C-terminal tails of U1-C and SmB/B' are highlighted (red and blue boxes respectively). Indicated are: U1 snRNA (magenta); Sm-B/B' (blue); Sm-D1, D2, D3, E, F and G (cyan); U1-70k (orange); U1-C (red); speculative path of the C-terminal tails of U1-70k isoforms 1 and 2 (dashed orange lines).

Mentions: One of the important insights gained from our study is the observation that U1-70k isoform 2 binds more stably to Sm-B/B' than isoform 1 while the converse is true for U1-C. This is an intriguing result given the close sequence similarity that exists between the two U1-70k isoforms, the only difference being an additional 9 amino acids (residues 223–231) for isoform 1. Interestingly however this sequence incorporates Ser226 shown in our proteomics experiments to be phosphorylated (figure S7) in line with previous reports [43], [46]. This therefore implies that the combination of the extra length of protein chain as well as the additional phosphorylation site enhances interactions with U1-C (figure 6). In contrast, the shorter version of U1-70k (isoform 2), with one less phosphorylation site, increases interactions with Sm-B/B'. It is established crystallographically that the N-terminus of U1-70k is extended and wraps around the ring to contact U1-C [17]. This path begins just N-terminal to its RDB, the motif that mediates its interaction with a stem-loop of U1 snRNA [17]. The additional amino acids in U1-70k isoform 1 occur C-terminal to the structured RBD and are also predicted to be unstructured [17]. Given our results that show that the C-terminal tails of the two U1-70k isoforms interact differently with Sm-B/B' and U1-C, on the opposite side of the ring to the extended N-terminus, and considering the high proportion of acidic residues in the C-terminal 45 residues (22% cf 4% basic) these unstructured acidic residues are poised for interaction with complementary basic groups. Given the highly basic arginine-rich C-terminal sequences of Sm-B/B' and U1-C and the fact that the C-terminus contains all but one of the six phosphorylation sites reported for U1-70k, it is entirely feasible that phosphorylation is responsible for fine tuning the interactions of the negatively charged C-terminal tails with the unstructured regions of positively charged Sm-B/B' and U1-C. This allows us to propose that an important role of U1-70k is in fine tuning interactions with either U1-C or Sm-/B/B' in response to incorporation of the different isoforms and their phosphorylation status in vivo.


Isoforms of U1-70k control subunit dynamics in the human spliceosomal U1 snRNP.

Hernández H, Makarova OV, Makarov EM, Morgner N, Muto Y, Krummel DP, Robinson CV - PLoS ONE (2009)

A model of interactions between the C-terminal tails of U1-70k isoforms 1 (left) and 2 (right) with U1-C and Sm-B/B', respectively.Possible path of the C-terminal tails of two U1-70k isoforms drawn approximately to scale (dashed curved orange lines) and modeled onto the crystal structure of human U1 snRNP [17] and interactions between C-terminal tails of U1-C and SmB/B' are highlighted (red and blue boxes respectively). Indicated are: U1 snRNA (magenta); Sm-B/B' (blue); Sm-D1, D2, D3, E, F and G (cyan); U1-70k (orange); U1-C (red); speculative path of the C-terminal tails of U1-70k isoforms 1 and 2 (dashed orange lines).
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Related In: Results  -  Collection

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pone-0007202-g006: A model of interactions between the C-terminal tails of U1-70k isoforms 1 (left) and 2 (right) with U1-C and Sm-B/B', respectively.Possible path of the C-terminal tails of two U1-70k isoforms drawn approximately to scale (dashed curved orange lines) and modeled onto the crystal structure of human U1 snRNP [17] and interactions between C-terminal tails of U1-C and SmB/B' are highlighted (red and blue boxes respectively). Indicated are: U1 snRNA (magenta); Sm-B/B' (blue); Sm-D1, D2, D3, E, F and G (cyan); U1-70k (orange); U1-C (red); speculative path of the C-terminal tails of U1-70k isoforms 1 and 2 (dashed orange lines).
Mentions: One of the important insights gained from our study is the observation that U1-70k isoform 2 binds more stably to Sm-B/B' than isoform 1 while the converse is true for U1-C. This is an intriguing result given the close sequence similarity that exists between the two U1-70k isoforms, the only difference being an additional 9 amino acids (residues 223–231) for isoform 1. Interestingly however this sequence incorporates Ser226 shown in our proteomics experiments to be phosphorylated (figure S7) in line with previous reports [43], [46]. This therefore implies that the combination of the extra length of protein chain as well as the additional phosphorylation site enhances interactions with U1-C (figure 6). In contrast, the shorter version of U1-70k (isoform 2), with one less phosphorylation site, increases interactions with Sm-B/B'. It is established crystallographically that the N-terminus of U1-70k is extended and wraps around the ring to contact U1-C [17]. This path begins just N-terminal to its RDB, the motif that mediates its interaction with a stem-loop of U1 snRNA [17]. The additional amino acids in U1-70k isoform 1 occur C-terminal to the structured RBD and are also predicted to be unstructured [17]. Given our results that show that the C-terminal tails of the two U1-70k isoforms interact differently with Sm-B/B' and U1-C, on the opposite side of the ring to the extended N-terminus, and considering the high proportion of acidic residues in the C-terminal 45 residues (22% cf 4% basic) these unstructured acidic residues are poised for interaction with complementary basic groups. Given the highly basic arginine-rich C-terminal sequences of Sm-B/B' and U1-C and the fact that the C-terminus contains all but one of the six phosphorylation sites reported for U1-70k, it is entirely feasible that phosphorylation is responsible for fine tuning the interactions of the negatively charged C-terminal tails with the unstructured regions of positively charged Sm-B/B' and U1-C. This allows us to propose that an important role of U1-70k is in fine tuning interactions with either U1-C or Sm-/B/B' in response to incorporation of the different isoforms and their phosphorylation status in vivo.

Bottom Line: Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle.Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo.These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
Most human protein-encoding genes contain multiple exons that are spliced together, frequently in alternative arrangements, by the spliceosome. It is established that U1 snRNP is an essential component of the spliceosome, in human consisting of RNA and ten proteins, several of which are post-translationally modified and exist as multiple isoforms. Unresolved and challenging to investigate are the effects of these post translational modifications on the dynamics, interactions and stability of the particle. Using mass spectrometry we investigate the composition and dynamics of the native human U1 snRNP and compare native and recombinant complexes to isolate the effects of various subunits and isoforms on the overall stability. Our data reveal differential incorporation of four protein isoforms and dynamic interactions of subunits U1-A, U1-C and Sm-B/B'. Results also show that unstructured post-translationally modified C-terminal tails are responsible for the dynamics of Sm-B/B' and U1-C and that their interactions with the Sm core are controlled by binding to different U1-70k isoforms and their phosphorylation status in vivo. These results therefore provide the important functional link between proteomics and structure as well as insight into the dynamic quaternary structure of the native U1 snRNP important for its function.

Show MeSH